title: Transfection of immortalized keratinocytes by low toxic poly(2-(dimethylamino)ethyl methacrylate)-based polymers.
abstract: Skin carcinoma are among the most spread diagnosed tumours in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based polymers, with original architecture and functionalities. We tested PDMAEMA polymers with different structures: linear, with two (DEA-PDMAEMA) or three (TEA-PDMAEMA) arms. The cytotoxicity of these polymers was assessed over a wide range of apparent M n (from 7600 to 64 600). At a N/P ratio of 7.38, cytotoxicity increases with the M n. Keratinocytes were transfected with a fluorescent oligonucleotide and then analyzed by flow cytometry. For the three architectures tested, the percentage of transfected cells and abundance of internalized oligonucleotide were closely related to the M n of the polymer. Confocal microscopy and FACS analyses showed a wide spread fine granular distribution of the oligonucleotide up to 3 days post-transfection. Then, we assessed the silencing efficiency of the polymers, targeting GFP in GFP expressing keratinocytes. The maximal silencing effect (±40%) was obtained using a DEA-PDMAEMA polymer (M n = 30 300). These results suggest that PDMAEMA-based polymers can be efficiently used to transfect immortalized keratinocytes and, thus, open new perspectives in the therapy of skin carcinoma.
authors.affiliation: a Research Unit of Cell Biology, University of Namur, B-5000 Namur, Belgium. nancy.vanoverstraeten@fundp.ac.be.
authors.forename: Nancy
authors.initials: N
authors.lastname: Van Overstraeten-Schlögel
authors.forename: Yong
authors.initials: Y
authors.lastname: Ho-Shim
authors.forename: Virginie
authors.initials: V
authors.lastname: Tevel
authors.forename: Sébastien
authors.initials: S
authors.lastname: Bontems
authors.forename: Philippe
authors.initials: P
authors.lastname: Dubois
authors.forename: Martine
authors.initials: M
authors.lastname: Raes
mesh_terms: D049109:Cell Proliferation; D005456:Fluorescent Dyes; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D008689:Methacrylates / Q000737:chemistry / Q000633:toxicity; D015394:Molecular Structure; D009757:Nylons / Q000737:chemistry / Q000633:toxicity; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012878:Skin Neoplasms / Q000235:genetics / Q000628:therapy; D014162:Transfection / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings; D008689:Methacrylates; D009757:Nylons; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C516130:hostasol; C407037:poly(2-(dimethylamino)ethyl methacrylate); D049452:Green Fluorescent Proteins